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. 2011 Mar;23(3):216-23.
doi: 10.1111/j.1365-2826.2010.02101.x.

Stress induces parallel changes in corticotrophin-releasing hormone (CRH) Transcription and nuclear translocation of transducer of regulated cAMP response element-binding activity 2 in hypothalamic CRH neurones

Y Liu  1 , H S Knobloch, V Grinevich, G Aguilera
Affiliations

Stress induces parallel changes in corticotrophin-releasing hormone (CRH) Transcription and nuclear translocation of transducer of regulated cAMP response element-binding activity 2 in hypothalamic CRH neurones

Y Liu et al. J Neuroendocrinol. 2011 Mar.

Abstract

Recent studies in vitro have shown that the cAMP response element-binding (CREB) co-activator, transducer of regulated CREB activity (TORC), is required for transcriptional activation of the corticotrophin-releasing hormone (CRH) gene. To determine the physiological importance of TORC2 regulating CRH transcription during stress, we examined the localisation of TORC2 in CRH neurones, as well as the relationship between changes in CRH heterogeneous nuclear (hn)RNA, nuclear translocation of TORC2 and binding of TORC2 to the CRH promoter. Immunohistochemistry revealed TORC2 immunoreactivity (irTORC2) in the dorsolateral (magnocellular) and dorsomedial (parvocellular) regions of the hypothalamic paraventricular nucleus (PVN). Although staining was mostly cytosolic under basal conditions, there was a marked increase in nuclear irTORC2 in the dorsomedial region after 30 min of restraint, concomitant with increases in CRH hnRNA levels. Levels of nuclear irTORC2 and CRH hnRNA had returned to basal 4 h after stress. Double-staining immunohistochemistry showed TORC2 co-staining in 100% of detected CRH neurones, and nuclear translocation after 30 min of restraint in 61%. Cellular distribution of TORC2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation experiments showed recruitment of TORC2 and phosphorylated CREB (pCREB) by the CRH promoter after 30 min of restraint, but 4 h after stress only pCREB was associated with the CRH promoter. The demonstration that TORC2 translocates to the nucleus of hypothalamic CRH neurones and interacts with the CRH promoter in conjunction with the activation of CRH transcription during restraint stress, provides strong evidence for the involvement of TORC2 in the physiological regulation of CRH transcription.

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Figures

Fig 1
Fig 1
Effect of restraint stress on CRH hnRNA, measured by in situ hybridization (A), and immunoreactive TORC 2 (irTORC 2), measured by western blot in nuclear and cytoplamic proteins in the microdissected dorsal hypothalamic region from individual rats (B). Rats were subjected to restraint stress for 1h and killed at 30 min or 4h after termination of the stress (240 min). Data are the mean and SE of data obtained from 5 rats for CRH hnRNA or 6 rats for irTORC 2. The intensity of the TORC 2 bands were corrected for β-actin in the cytoplasm and for HDAC1 in the nucleus. Images from representative rats are shown above the bars. *, p< 0.05; **, p< 0.001
Fig 2
Fig 2
Immunohistochemical staining for TORC 2 in hypothalamic sections from control rats (basal), 30 min restraint and 3h after 1h restraint (240 min) (A). As shown by the higher magnification of representative images in the lower panels, there is an increase in nuclear staining for TORC 2 following 30 min restraint. Replacing the TORC 2 antibody by non-immune globulin yielded no staining over the background. The quantitative analysis of the number of cells showing nuclear and cytoplasmic staining in the dorsomedial and dorsolateral regions of the PVN is shown in B. Bars represent the mean and SE of measurements expressed as percent of cytosolic and nuclear location per total number of cells were counted in each animal in 3 rats. *, p<0.01 higher than nuclear staining in basal (time 0), or 3h after termination of 1h stress (240 min). #, p<0.01 lower than cytoplasmic staining in basal, or 3h after termination of 1h stress (240 min).
Fig 3
Fig 3
Co-localization of immunoreactive CRH and immunoreactive TORC 2 in the dorsomedial region of the PVN Rats were subjected to restraint stress for up to 1h and killed at 30 min or 3h after termination of the stress (240 min) (A). Hypothalamic sections were stained for TORC 2 and CRH as described in Methods. CRH neurons with nuclear localization of irTORC2 indicated by arrows, and neurons with cytoplasmic irTORC 2 are shown by arrowheads. Nuclear localization of TORC2 in rats subjected to 30 min restraint was confirmed by DAPI staining as shown in B. Arrows point to the corresponding DAPI staining in 4 neurons. No staining was observed in the absence of TORC and CRH antibodies. The magnification bars correspond to 20μm.
Fig 4
Fig 4
Restraint stress induces recruitment of phospho-CREB and TORC2 by the CRH promoter in the hypothalamic PVN region. ChIP assays using phospho-CREB and TORC 2 antibodies and cross-linked DNA from microdissected hypothalamic PVN region of control rats and rats subjected to restraint stress for 30min or 3h after 1h restraint (240 min) (A). Amounts of CRH promoter DNA in the immunoprecipitant was measured by qRT-PCR. Bars represent the mean and SE of the results obtained in 4 experiments using pooled tissue from 2 or 3 rats per experimental point. Gel shows the results obtained in pools of hypothalamic tissue from 3 rats per group in a representative experiment (B). **, p< 0.01 higher than basal; **, p<0.02 higher than basal; #, p<0.02 lower than 30 min.
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